Approximately half of traumatic spinal cord injury (SCI) cases affect cervical spinal cord regions, resulting in debilitating and often chronic respiratory compromise. The majority of these injuries affect mid-cervical spinal cord levels, the location of the important pool of phrenic motor neurons (PMNs) that innervates the diaphragm, the primary muscle of inspiration. Following initial trauma to cervical spinal cord, a valuable opportunity exists for preventing secondary PMN degeneration and consequently preserving respiratory function. One of the major causes of secondary injury following SCI is excitotoxic cell death due to dysregulation of extracellular glutamate homeostasis. In the central nervous system (CNS), glutamate is efficiently cleared from the synapse and other sites by glutamate transporters. Astrocytes are supportive glial cells that play a host of crucial roles in CNS function. In particular, astrocytes express the major CNS glutamate transporter, GLT1, which is responsible for the vast majority of functional glutamate uptake in most CNS regions, particularly spinal cord. Preliminary findings from our lab show that: 1) levels of intraspinal GLT1 expression and GLT1-mediated glutamate uptake are reduced in an animal model of cervical contusion SCI; 2) histological and functional outcomes following SCI are worsened in GLT1 heterozygous mice; 3) increasing intraspinal GLT1 levels via injection of AAV1-GLT1 viral vector decreases PMN loss and diaphragm dysfunction after cervical contusion; 4) intraspinal astrocyte transplantation decreases secondary degeneration after thoracic contusion, and transplantation of astrocytes engineered to constitutively overexpress GLT1 further enhances efficacy. Proposed studies will test the central hypothesis that astrocyte GLT1 loss plays a key role in secondary respiratory PMN degeneration. With the goal of developing a viable therapy for SCI patients, studies will test intraspinal transplantation of a clinically-relevant source of cells, human induced Pluripotent Stem (iPS) cell- derived astrocytes (hIPSAs), in a cervical contusion model. By targeting GLT1, this stem cell-based astrocyte replacement strategy aims to protect PMNs from glutamate excitotoxicity during secondary degeneration. As therapeutic efficacy is a function of transplant integration in diseased CNS, studies in Aim #1 will characterize in vivo survival, differentiation and long-term safety of hIPSAs following intraspinal transplantation in a mouse model of cervical contusion SCI. As GLT1 is a promising target for transplant-based astrocyte replacement in SCI, studies in Aim #2 will examine in vivo ability of transplanted hIPSAs to express GLT1 and to increase intraspinal GLT1 protein and glutamate uptake levels after cervical contusion. Results will show whether, similar to endogenous astrocytes after contusion SCI, transplanted hIPSAs have reduced propensity for GLT1 expression and function, which has important relevance for their therapeutic potential in SCI. hIPSAs will also be engineered to constitutively overexpress GLT1 to enhance therapeutic potential. In Aim #3, studies will evaluate in vivo efficacy of hIPSAs for PMN protection and consequent preservation of diaphragm function.